Octavio E. Sousa

Phylogeography and Genetic Variation of Triatoma dimidiata, the Main Chagas Disease Vector in Central America, and Its Position within the Genus Triatoma

Background Among Chagas disease triatomine vectors, the largest genus, Triatoma, includes species of high public health interest. Triatoma dimidiata, the main vector throughout Central America and up to Ecuador, presents extensive phenotypic, genotypic, and behavioral diversity in sylvatic, peridomestic and domestic habitats, and non-domiciliated populations acting as reinfestation sources. DNA sequence analyses, phylogenetic reconstruction methods, and genetic variation approaches are combined to investigate the haplotype profiling, genetic polymorphism, phylogeography, and evolutionary trends of T. dimidiata and its closest relatives within Triatoma. This is the largest interpopulational analysis performed on a triatomine species so far. Methodology and Findings Triatomines from Mexico, Guatemala, Honduras, Nicaragua, Panama, Cuba, Colombia, Ecuador, and Brazil were used. Triatoma dimidiata populations follow different evolutionary divergences in which geographical isolation appears to have had an important influence. A southern Mexican–northern Guatemalan ancestral form gave rise to two main clades. One clade remained confined to the Yucatan peninsula and northern parts of Chiapas State, Guatemala, and Honduras, with extant descendants deserving specific status. Within the second clade, extant subspecies diversity was shaped by adaptive radiation derived from Guatemalan ancestral populations. Central American populations correspond to subspecies T. d. dimidiata. A southern spread into Panama and Colombia gave the T. d. capitata forms, and a northwestern spread rising from Guatemala into Mexico gave the T. d. maculipennis forms. Triatoma hegneri appears as a subspecific insular form. Conclusions The comparison with very numerous Triatoma species allows us to reach highly supported conclusions not only about T. dimidiata, but also on different, important Triatoma species groupings and their evolution. The very large intraspecific genetic variability found in T. dimidiata sensu lato has never been detected in a triatomine species before. The distinction between the five different taxa furnishes a new frame for future analyses of the different vector transmission capacities and epidemiological characteristics of Chagas disease. Results indicate that T. dimidiata will offer problems for control, although dwelling insecticide spraying might be successful against introduced populations in Ecuador.

Trypanosoma rangeli. Epimastigote Immunogenicity and Cross-Reaction with Trypanosoma cruzi

Trypanosoma rangeli epimastigote components, able to elicit an immune response, were defined and compared with those found in Trypanosoma cruzi epimastigotes. Using polyclonal antibodies against these parasites and immunoblotting analysis, an antigenic similarity index of 0.48 was found when anti-T. cruzi antibodies were used and 0.60 with anti-T. rangeli antibodies. Additionally, immunoblotting analyses using specific antibodies against a T. rangeli polypeptide eluted from polyacrylamide gels confirm an antigen of 43 kDa as a specific marker for T. rangeli.

Serological Diagnosis of Trypanosoma rangeli Infected Patients. A Comparison of Different Methods and its Implications for the Diagnosis of Chagas' Disease

Venous blood from 65 Panamanian schoolchildren living in an area endemic for both Trypanosoma cruzi and T. rangeli were screened for the presence of these parasites. Trypanosoma rangeli were isolated and cultured from four individuals. Serological tests of all 65 sera were performed, including immunohaemagglutination (IHA), indirect immunofluorescence assay (IF) and ELISA using both T. rangeli and T. cruzi as antigens, as well as T. cruzi synthetic peptides in an ELISA assay. Results indicated a higher immunoreactivity to T. rangeli preparations than to T. cruzi within the studied population, which could be divided into four ‘serological responder’ groups. Interestingly, the panel of SAPA and other T. cruzi synthetic peptides were not useful in the discrimination of patients. Furthermore, patients from whom parasites had been isolated could not be distinguished from those of two other groups. Significant immunoreactivityto T. cruzi preparations was displayed in all responder sera, despite total lack of evidence of infection with this parasite. The immunobiological significance of T. rangeli infection is unclear, but these data indicate that it is a compounding problem in the accurate diagnosis of pathological T. cruzi infection by serological analysis. The relationship of these cohabiting species, in respect to infection outcome and immunological activation, is discussed.

Molecular characterization of human Trypanosoma cruzi isolates from endemic areas in Panama

The present work provides information on Trypanosoma cruzi genotype circulating in endemic areas of Chagas disease in Panama. A total of 26 crude stocks of T. cruzi, isolated from the blood of persons with different clinical profiles of Chagas disease were collected and crio-conserved until used. Most of the stocks had been characterized by means of isoenzyme electrophoresis on cellulose acetate membranes. The clinical profiles of infected persons included 9 (34.6%) asymptomatic and 17 acute (65.4%) including 5 (19.2%) fatal cases, 2 under 5 years old and 3 adults. A multiplex-PCR assay based on the amplification of the non-transcribed spacer of the mini-exon gene was performed. All stocks of T. cruzi included in the study were found to correspond to Tc I group. This result supports the predominance of T. cruzi-I in the transmission cycles affecting the human population in the Republic of Panama.

Trypanosoma rangeli and Trypanosoma cruzi: cross-reaction among their immunogenic components

It has been demonstrated that immuno-modu-lating mechanisms induced by epimastigotes ofTrypanosoma rangeli, an assumed harmless hu-man parasite, may have relevance to protectionagainst Trypanosoma cruzi, the causative organ-ism of Chagas’ disease (B Basso et al. 1991 Am JTrop Med Hyg 44: 413-419). Evidently, this find-ing is related to the previously demonstrated an-tigenic similarity, between these parasites (DAfchain et al. 1979 J Parasitol 65: 507-514, BBasso et al. 1989 Rev Lat-amer Microbiol 31 : 141-146, M Grogl, RE Kuhn 1984 J Parasitol 70 : 822-824, F Guhl, CJ Marinkelle 1982 Ana Trop MedParasitol 76: 361). However, at the present timeit is not known which T. rangeli antigens can elicitantibodies capable to recognize T. cruzi compo-nents. In order to clarify this matter, we used theimmunoblotting technique to delineate the cross-reactivity among the immunogenic components ofthese parasites.The parasites used were isolated from humansin central Panama. Clones derived from single cellisolates (JA Dvorak 1985, Rev Soc Bras Med Trop18 (Suppl): 29-38) of T. rangeli (LMCL2) and T.cruzi (MA-081A) were characterized by studiesof morphology, intracellular multiplication mea-sured in vitro and infectivity for vector salivaryglands. Flagellates were cultivated and lyophilizedfollowing procedures designed previously (NHVattuone, JF Yanovsky 1971 Exp Parasitol 30:349-355, A Saldana 1990 ImmunoparasitologicalStudies of Trypanosoma cruzi clones from Panama ,MSc Thesis, I. Karolinska-Stockholm, 90 pp). Thelyophilized T. rangeli and T. cruzi epimastigoteswere used in the production of mouse antibodiesas ascitic fluid (AS Tung et al. 1976 J Immunol116: 676-681, AE Horna 1992 Biochemical andImmunological Characterization of Trypanosomarangeli (Tejera 1920) strains affecting rural popu-lations of Central and South America. MSc The-sis, I. Karolinska-Stockholm, 100 pp). To demon-strate the antibody cross-reactivity we followedprocedures described earlier (EC Rostjord et al.1990 J Parasitol 76: 698-702).Previous reports suggested that exposure to T.rangeli antigens might modify the pathology dueto T. cruzi (Grogl, Kuhn loc. cit., F Guhl et al.1987 Parasitol 94: 475-484, L Hudson et al. 1988Parasitol 96: 449-460). Nevertheless, as far as weknow , the first work that demonstrated a partialresistance in T. rangeli-immunized mice againstT. cruzi infection was done by Basso et al. (loc.cit.). However, the studies of T. rangeli immuno-genic components seems to be just beginning.Our results (Figs 1, 2) revealed that there areseveral common epitopes among T. rangeli anti-gens and between T. rangeli and T. cruzi polypep-tides. The antibodies eluted from the regions A,B, C, D, E and F recognized additional bands tothe bands from which they were separated eitherin T. rangeli or T. cruzi immunoblotting profiles.Most of them cross-reacted with antigens in re-gion B ( 81, 76 and 71 Kda). However, when theantibodies eluted from regions of 34 (G), 29 (H)and 24 Kda (I) were used, more specificity wasfound. The antibodies from the bands of 34 and29 Kda recognized, without differences, antigenswith similar molecular weights in the T. rangelior T. cruzi profiles. This may, therefore, representthe expression of two antigens, highly conserved,which apparently are a second group ofimmunodominant determinants responsible for theobserved cross-reactivity.An additional finding was the specific recog-nition pattern noted with the antibodies from the24 Kda region, apparently this antigen shows athird restrictive group of immunodominantepitopes, expressed either in the 24 Kda polypep-tide of T. rangeli and in the 23 Kda region of T.cruzi.Even with these cross-reactions, it should bekept in mind, that it is possible to find specificepitopes in each one of these molecules. The iden-tification of specific epitopes recognized by mono-clonal antibodies should facilitate studies whichaim at settling this possibility.Finally, research on purification and immu-nochemistry of these T. rangeli antigens, whichcross-reacted with T. cruzi components, could beimportant on the diagnostic and management ofChagas’ disease.

Genotyping of Panamanian Trypanosoma cruzi stocks using the calmodulin 3'UTR polymorphisms

Mutations in the 3′ untranslated region of calmodulin gene have recently been reported to be specific to different Trypanosoma cruzi lineages. In the present report, this molecular marker was used to genotype 24 T. cruzi isolates from humans and vectors from different endemic areas in Panama. The finding of solely T. cruzi I genotype might explain the low morbidity of Chagas’ disease in the region when compared to other countries in Latin America.

AFLP Polymorphisms Allow High Resolution Genetic Analysis of American Tegumentary Leishmaniasis Agents Circulating in Panama and Other Members of the Leishmania Genus

American Tegumentary Leishmaniasis is caused by parasites of the genus Leishmania, and causes significant health problems throughout the Americas. In Panama, Leishmania parasites are endemic, causing thousands of new cases every year, mostly of the cutaneous form. In the last years, the burden of the disease has increased, coincident with increasing disturbances in its natural sylvatic environments. The study of genetic variation in parasites is important for a better understanding of the biology, population genetics, and ultimately the evolution and epidemiology of these organisms. Very few attempts have been made to characterize genetic polymorphisms of parasites isolated from Panamanian patients of cutaneous leishmaniasis. Here we present data on the genetic variability of local isolates of Leishmania, as well as specimens from several other species, by means of Amplified Fragment Length Polymorphisms (AFLP), a technique seldom used to study genetic makeup of parasites. We demonstrate that this technique allows detection of very high levels of genetic variability in local isolates of Leishmania panamensis in a highly reproducible manner. The analysis of AFLP fingerprints generated by unique selective primer combinations in L. panamensis suggests a predominant clonal mode of reproduction. Using fluorescently labeled primers, many taxon-specific fragments were identified which may show potential as species diagnostic fragments. The AFLP permitted a high resolution genetic analysis of the Leishmania genus, clearly separating certain groups among L. panamensis specimens and highly related species such as L. panamensis and L. guyanensis. The phylogenetic networks reconstructed from our AFLP data are congruent with established taxonomy for the genus Leishmania, even when using single selective primer combinations. Results of this study demonstrate that AFLP polymorphisms can be informative for genetic characterization in Leishmania parasites, at both intra and inter-specific levels.

TRYPANOSOMA RANGELI: IDENTIFICATION AND PURIFICATION OF A 48-KDA-SPECIFIC ANTIGEN

Trypanosoma rangeli infects humans nonpathologically in some areas of Central and South America. Due to morphological and antigenic similarities with T. cruzi, the clear identification of this parasite is an important task. Here, we describe the identification and purification of a specific 48-kDa antigen from T. rangeli. By western blotting analysis, this molecule was not detected in T. cruzi epimastigotes and in Leishmania sp. promastigotes. Fluorescence-activated cell sorter analysis demonstrated that the protein is expressed uniformly by the T. rangeli cell population during axenic culture. Additionally, following immunostaining, a particular subcellular localization (present in organelles) is proposed for this antigen. These results suggest that this 48-kDa antigen may be a useful marker for the identification and characterization of T. rangeli isolates.

Trypanosoma rangeli sialidase: kinetics of release and antigenic characterization

The epimastigote stage of Trypanosoma rangeli release a sialidase with a high sialic acid hydrolysis capacity. We demonstrate that sialidase secretion is an active process that is reduced at low temperatures and in the presence of sodium azide. The enzyme is continuously released until certain maximally active concentrations are attained in the BHI culture medium when the parasite density reaches 2-3 x 10(6) cells. When introduced into culture medium already containing such enzyme levels, freshly harvested parasites do not secrete additional sialidase. These findings suggest a self-regulating mechanism and a biological role for the secreted T. rangeli sialidase. The secreted enzyme was purified to homogeneity by fractionation with ammonium sulphate and affinity chromatography. Antibodies raised against the purified molecule recognized antigens of similar molecular weights (73 kDa) in western immunoblotting analyses of T. rangeli and T. cruzi whole cell lysates. No antigenic recognition was recorded against T. cruzi active sialidase/trans-sialidase polypeptides or Clostridium perfringens and Vibrio cholerae commercial sialidases. These observations may indicate the expression of different antigenic domains in T. rangeli, T. cruzi and bacterial sialidases.

Trypanosoma rangeli sialidase: lack of activity in stocks from Panama and other regions

A total of 33 crude and cloned Trypanosoma rangeli stocks found as natural infections in human from Panama and other endemic areas of Central and South America were evaluated as producers of sialidase (SA) activity through the MU-NANA fluorescence test. Negative results were observed in 6 of the isolates: Panama (4), Honduras (1), and Brazil (1). In addition, an immunoblotting analysis confirm the presence of the SA antigen in these stocks without enzymatic activity. These findings must be considered in the interpretation of the biological significance of T. rangeli SA and in the proper characterization and identification of this parasite.